The present invention relates generally to molecules such as peptides, polypeptides and proteins which interact immunologically with antibodies or T-cells in subjects having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM). These molecules are preferentially immunoreactive to T-cells in subjects having pre-clinical or clinical IDDM and are useful in the development of diagnostic, therapeutic and prophylactic agents for IDDM.
Amino acid sequences are referred to herein by sequence identity numbers (SEQ ID NOs) which are defined at the end of the specification.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers, but not to the exclusion of any other element or integer or group of elements or integers.
Insulin-Dependent Diabetes Mellitus is a serious disease resulting from the destruction of insulin-secreting xcex2-cells, probably mediated by T cells that recognise xcex2-cell autoantigens. A major antigen implicated in T-cell mediated xcex2-cell destruction characteristic of IDDM is glutamic acid decarboxylase (GAD), which occurs in two major isoforms, GAD 65 and GAD 67. These two isoforms have approximately 65% similarity at the amino acid sequence level. Subjects with IDDM or at high-risk of the disease show autoantibody and autoreactive T-cell responses to GAD insulin or both autoantigens. In NOD mice, an animal model for spontaneous IDDM, GAD is a dominant and early target antigen (Tisch et al Nature 366:72-75, 1993).
Identification of the immunodominant epitope(s) of pathogenic autoantigens involved in xcex2-cell autoimmunity could lead to improved methods of diagnosis as well as therapeutic strategies to prevent IDDM.
In work leading up to the present invention, the inventors sought to identify immunodominant epitopes in GAD and proinsulin molecules in order to improve upon current diagnostic procedures and to further develop therapeutic and prophylactic compositions and treatment approaches for IDDM.
In accordance with the present invention, peptides were synthesised based on a thirteen amino acid region of high similarity between the sequences of human GAD 65 (amino acid residue numbers 506-518) and human proinsulin (amino acid residue numbers 24-36), which region of similarity also extends to human GAD 67 and mouse proinsulins and mouse GADs (FIG. 1). The immunoreactivity of these peptides is identified in accordance with the present invention on the basis of interactivity of peripheral blood cells or T-cells obtained from the peripheral blood of subjects with pre-clinical or clinical IDDM, thereby forming the basis for a new range of diagnostic, therapeutic and prophylactic procedures for IDDM.
Accordingly, one aspect of the present invention provides a recombinant or synthetic peptide or chemical equivalents thereof of the formula:
X1X2X3
wherein:
X1 and X3 may be the same or different and each is an amino acid sequence comprising from 0 to 40 naturally or non-naturally occurring amino acid residues;
X2 is any amino acid sequence of from 10 to 100 residues derived from, homologous to or contiguous within amino acids 506 to 518 inclusive or derivatives thereof of human GAD65 and/or amino acids 24 to 36 inclusive or derivatives thereof of human proinsulin; and wherein said peptide molecule is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM). Preferred cells include but are not limited to peripheral blood mononuclear cells (PBMCs), anticoagulated whole blood and tissue biopsy cells.
Reference to a xe2x80x9cpeptidexe2x80x9d includes reference to a polypeptide or protein or parts thereof.
In a preferred embodiment X2 comprises not less than about 10 and not greater than about 50, amino acid residues, more preferably not less than about 10 and not greater than about 30 amino acid residues and even more preferably not less than about 10 and not greater than about 15.
In a particularly preferred embodiment X2 has either of the following amino acid sequences:
F F Y T P K T R R E A E Dxe2x80x83xe2x80x83[SEQ ID NO:1];
or
F W Y I P P S L R T L E Dxe2x80x83xe2x80x83[SEQ ID NO:2].
According to this preferred embodiment, there is provided a recombinant or synthetic peptide or chemical equivalent thereof comprising the sequence:
X1X2X3
wherein
X1 and X3 may be the same or different and each is an amino acid sequence comprising from 0 to 15 naturally or non-naturally occurring amino acid residues;
X2 is selected from FFYTPKTRREAED and FWYIPPSLRTLED or a derivative or chemical equivalent thereof and wherein said peptide is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects with pre-clinical or clinical IDDM and determining reactivity by an appropriate assay. Preferred cells include but are not limited PBMCs, anti-coagulated whole blood or tissue biopsy cells and determining reactivity by an appropriate assay.
The peptides of the present invention may be prepared by recombinant or chemically synthetic means. According to a preferred aspect of the present invention, there is provided a recombinant peptide which is preferentially immunologically reactive with T-cells from individuals with clinical or pre-clinical IDDM, which is prepared by the expression of a host cell transformed with a cassette coding for the peptide sequences of the present invention. The peptide may be fused to another peptide, polypeptide or protein. Alternatively, the peptide may be prepared by chemical synthetic techniques, such as by the Merrifield solid-phase synthesis procedure. The synthetic or recombinant peptide may or may not retain GAD activity or proinsulin activity. Furthermore, although synthetic peptides of the formula given above represent a preferred embodiment, the present invention also extends to biologically pure preparations of the naturally occurring peptides or fragments thereof. By xe2x80x9cbiologically purexe2x80x9d is meant a preparation comprising at least about 60%, preferably at least about 70%, more preferably at least about 80% and still more preferably at least about 90% or greater as determined by weight, activity or other suitable means.
By xe2x80x9cpre-clinical IDDMxe2x80x9d as used herein means those subjects who may or may not be first degree relatives of someone with IDDM who have genetic and/or immune markers of pancreatic islet (xcex2) cell autoimmunity. By xe2x80x9cimmune markersxe2x80x9d is meant amongst other parameters known to those in the art to include circulating antibodies and/or T-cells reactive with islet (xcex2) cell autoantigens.
By xe2x80x9cderivativesxe2x80x9d as used herein is taken to include any single or multiple amino acid substitution, deletion and/or addition relative to the naturally occurring amino acid sequence in the native molecule from which the peptide is derived including any single or multiple substitution, deletion and/or addition of other molecules associated with the peptide, including carbohydrate, lipid and/or other proteinacious moieties. Such derivatives, therefore, include glycosylated or non-glycosylated forms or molecules with altered glycosylation patterns.
By the term xe2x80x9creacting with T cells and modifying T-cell functionxe2x80x9d as used herein is taken to include T-cell activation, T-cell inactivation and/or T-cell death.
The present invention also covers chemical analogues of the subject peptides which include, but is not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives, during peptide synthesis and the use of cross-linkers and other methods which impose conformational constraints on the peptides or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5xe2x80x2-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Cxcex1 and Nxcex1-methylamino acids, introduction of double bonds between Cxcex1 and Cxcex2 atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The invention also extends to use of the peptides, or derivatives thereof of the present invention in the treatment of patients. In this latter aspect, such methods of treatment include their use as an adsorbent to remove autoantibodies or autoreactive cells from a patient, their use in direct administration to a patient as a means of desensitising or inducing immunological tolerance or other mechanisms to eliminate or diminish reactivity of autoreactive T-cells or autoantibodies to IDDM autoantigens or to generate T-cell lines or clones to be used for or as therapeutic agents.
According to this aspect of the present invention, there is provided a method of treatment comprising administering to a subject an effective amount of a peptide or chemical equivalent thereof for a time and under conditions sufficient to remove or substantially reduce the presence or function in said subject of autoreactive T-cells and/or autoantibodies to IDDM autoantigens wherein the peptide comprises the formula:
X1X2X3
wherein:
X1 and X3 may be the same or different and each is an amino acid sequence comprising from 0 to 40 naturally or non-naturally occurring amino acid residues;
X2 is any amino acid sequence of from 10 to 100 residues derived from, homologous to or contiguous within amino acids 506 to 518 inclusive or derivatives thereof of human GAD65 and/or amino acids 24 to 36 inclusive or derivatives thereof of human proinsulin; and wherein said peptide molecule is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects having clinical or pre-clinical Insulin-Dependent Diabetes Mellitus (IDDM). Preferred cells include but are not limited to peripheral blood mononuclear cells (PBMCs), anticoagulated whole blood and tissue biopsy cells.
The method of treatment contemplated herein includes, but is not limited to, the following examples. A first example of treatment is desensitisation or tolerance induction using an effective amount of synthetic peptide or derivative thereof to alter T-cell recognition of or response to GAD and/or pro-insulin and/or other IDDM antigens and/or induce T-cell suppression or regulation. This may be achieved by using the known effect of certain ultraviolet wavelengths, especially UV-B, to modify antigen presentation through the skin or transmucosal or systemic administration. Effective amounts of the peptides or derivatives thereof would be applied epicutaneously to the skin of subjects exhibiting peripheral blood T-cell reactivity to GAD or proinsulin peptides or polypeptides. After exposure of skin to UV-B radiation, treatment would be repeated until such time that T-cell reactivity to GAD or proinsulin was suppressed.
A second example of treatment is to induce mucosal-mediated tolerance using an effective amount of the subject peptides or derivatives thereof to alter T-cell recognition of or response to GAD and/or pro-insulin and/or other IDDM antigens and/or induce T-cell suppression using an effective amount of peptide or derivative thereof to alter T-cell recognition of or response to GAD and/or pro-insulin and/or other IDDM antigens and/or induce T-cell suppression by the administration of the peptide or derivatives thereof by oral, aerosol or intranasal means amongst other routes of mucosal administration.
Another treatment involves application of the subject peptides to the skin together with one or more cytokines such as but not limited to TNFxcex1 or xcex2. A further treatment involves systemic administration of soluble peptide via subcutaneous or intravenous routes to induce immunological tolerance. Yet another treatment involves T-cell immunisation whereby T-cell lines are generated to GAD or proinsulin peptide or polypeptide or fragments thereof by standard procedures, cells attenuated by fixation with agents such as glutaraldehyde or paraformaldehyde, washed under sterile conditions and re-injected into patients for a time and under conditions to cause suppression of the endogenous T-cell response to autoantigens. These approaches are applicable to the prevention of IDDM progression in asymptomatic subjects with pre-clinical IDDM or subjects with recentxe2x80x94onset clinical IDDM, as well as to the recurrence of IDDM in subjects who have received pancreas, islet cell or insulin-producing cell transplants. These approaches are also applicable to Stiff Man Syndrome (SMS) and other diseases where GAD and/or proinsulin is an autoantigen.
In accordance with the present invention, the effective amount of peptide is 0.1 xcexcg to 10 mg per dose and preferably 1.0 xcexcg to 1 mg per dose. A dose may comprise a single administration or protocol comprising single or multiple administration hourly, daily, weekly or monthly or at other suitable times. Administration may be by any convenient means such as, but not limited to, intravenous, subcutaneous, epicutaneous, infusion, oral, topical, intranansal, aerosol suppository or intraperitoneal administration. The peptide may be administered alone or in combination with one or more other active molecules such as molecules which facilitate the activity or action of the peptide for example lipopolysaccharide (LPS), choleratoxin xcex2-chain, Lymphocyte Functional Associated Antigen-3 (LFA-3), other adjuvants and in particular, tumour necrosis factor xcex1 (TNF-xcex1), tumour necrosis factor xcex2 (TNF-xcex2) or leukaemia inhibitory factor (LIF).
In yet a further embodiment, the present invention contemplates the use of the peptides described herein to measure reactivity of a subject""s cells to the IDDM autoantigen. The peptides or derivatives thereof may be added in solution or bound to a solid support together with cells derived from peripheral blood or from tissue biopsies either unfractionated, fractionated or derived as continuous cell lines. Reactivity to the autoantigen may then be measured by standard proliferation assays such as incorporation of tritiated thymidine, standard cytotoxic assays such as release of marker radioactivity from target cells, measurements of expressed or secreted molecules such as surface markers, cytokines or other standard assays of cellular reactivity which are well known in the art.
According to this aspect of the present invention, there is provided a method of assaying the reactivity of a subject to IDDM autoantigen, said method comprising contacting a peptide or chemical equivalent thereof comprising the formula:
X1X2X3
wherein:
X1 and X3 may be the same or different and each is an amino acid sequence comprising from 0 to 40 naturally or non-naturally occurring amino acid residues;
X2 is any amino acid sequence of from 10 to 100 residues derived from, homologous to or contiguous within amino acids 506 to 518 inclusive or derivatives thereof of human GAD65 and/or amino acids 24 to 36 inclusive or derivatives thereof of human proinsulin; and wherein said peptide molecule is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM) and determining reactivity by appropriate assay. In accordance with this assay, any cell type may be used but is preferably selected from PBMC""s, anti-coagulated whole blood cells or tissue biopsy cells.
Preferably, the present invention contemplates a method of assaying the reactivity of a subject to IDDM autoantigen said method comprising contacting a peptide or chemical equivalent thereof comprising the formula:
X1X2X3
wherein
X1 and X3 may be the same or different and each is an amino acid sequence comprising from 0 to 15 naturally or non-naturally occurring amino acid residues;
X2 is selected from FFYTPKTRREAED and FWYIPPSLRTLED or a derivative or chemical equivalent thereof and wherein said peptide is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects with pre-clinical or clinical IDDM and determining reactivity by an appropriate assay. Preferably, cells include but are not limited to peripheral blood mononuclear cells (PBMCs), anticoagulated whole blood and tissue biopsy cells.
In another embodiment of the present invention, there is provided a diagnostic kit for assaying T cells. Standard 96-well plates, as used in ELISA, are pre-coated with a monoclonal antibody (MAb) to a T-cell cytokine such as xcex3-interferon (xcex3-IFN) with or without antigen. Alternatively, antigen is added in soluble form together with aliquots of peripheral blood, peripheral blood mononuclear cells or T-cells. Incubation is allowed to proceed for one or more days, the supernatant (comprising medium and plasma) and the cells are washed off, wells washed again and plates developed with a labelled second MAb to the cytokine such as anti-xcex3-IFN conjugated with alkaline phosphatase or horseradish peroxidase. Colorimetric reaction and read-out systems can then be utilised. Alternatively, soluble cytokines (eg: xcex3-IFN) are measured in the supernatant by standard assays such as ELISA; further it is possible to visualise microscopically by the ELISPOT technique individual spots on bottoms of wells representing cytokine produced at the single cell level thereby enabling the frequency of peptide- epitope-reactive T-cells to be determined.
The present invention will now be further described with reference to the following non-limiting Figures and Examples.